Mca yvadapk dnp oh es007

HEK293 cells and HEK293 cells stably expressing murine iRhom2⁴² were transiently transfected with wtACE2_pMOWSCMV as described above. At a confluency of 90% in a 24‐well plate cells were treated with stimulators and inhibitors as described above for 2 h at 37°C. Supernatant was harvested and cell were lysed in ACE2 activity assay lysis buffer (50 mM Tris; 1% Triton X‐100; 137 mM NaCl; 2 mM EDTA; 5 mM ZnCl₂; 1 tablet cOmplete protease inhibitor; pH7.5). Assay solution contains 45 µl reaction buffer (50 mM Tris; 300 mM NaCl; 0.1 mM ZnCl₂; 0.01% Triton X‐100; adjust pH6.5) and 5 µl 0.1 mM fluorogenic ACE2 peptide substrate (Mca‐YVADAPK(Dnp)‐OH; ES007, R&D systems, Minneapolis, Minnesota, USA) in DMSO. 50 µl supernatant or 5 µl cell lysate were mixed with 50 µl assay solution and increase in fluorescence was continuously measured (ex: 320 nm; em: 405 nm) with the FLUOstar Optima. The relative ACE2 activity was calculated as substrate turnover (slope of the fluorescence increase) in the supernatant in relation to the total turnover in supernatant plus cell lysate (total amount of available substrate).